1. Field of the Invention
This invention is directed to a method. More specifically, this invention concerns a unique process for the rapid preparation of particulate analytes, notably cells, cell organelles and the like, for analysis by photooptical means. This process is particularly well suited for analysis of cellular analytes which are present in small concentrations and/or where a change in their relative proportion to another cell population is diagnostically significant. The ability of this process to afford such advantage over the more traditional techniques is based upon the conservative nature of the preparation process. The term "conservative" as used herein is intended as descriptive of the ability of this process to preserve virtually all of the blood components, with the exception of erythrocytes, for later analysis. The integrity of the sample, with respect to the conserved components, also provides the first truly reliable means for the establishment of standards against which other samples can be measured. This process is particularly well adapted to analysis of aged samples of whole blood, and to blood samples from disease state patients, wherein cell integrity is difficult to maintain and thus an accurate analysis heretofore impossible. This process is particularly well suited for rapid preparation of whole blood samples for analysis by flow cytometry.
2. Description of the Prior Art
The traditional techniques for the isolation and staining of the leukocyte fraction of whole blood has typically involved numerous physical manipulations of the sample and an inordinate number of wash steps. As has been appreciated and reported in the technical literature, these traditional techniques inherently result in some finite depletion of the leukocyte population from the sample; at least some alteration in the gross morphology of the leukocyte population which remain in the sample; at least some alteration in the markers on the surface of the leukocyte population; and, at least some displacement of the stain on the surface of the cell population of interest.
The techniques which have been used up to now in the preparation of leukocytes from whole blood samples for subsequent analysis, have traditionally involved: (1) the preparation of a buffy coat fraction by controlled centrifugation of the whole blood sample, followed by staining with a fluorochrome labelled conjugate; (2) concurrent treatment of a whole blood sample with a lytic reagent and a fluorochrome labelled conjugate; or (3) the preparation of a buffy coat fraction by controlled centrifugation of the whole blood sample and thereafter subjecting the buffy coat sample to further enrichment by Ficoll-Paque density gradient centrifugation. Such enrichment permits the recovery of an interfacial layer containing enriched mononuclear cells which are thereafter stained with a fluorochrome labelled conjugate.
Each of the above preparative procedures is labor intensive, requires repeated physical manipulation of the fraction containing the leukocytes of interest and is time consuming. Where conventional centrifugation techniques are used to obtain an enriched leukocyte sample, cell loss will invariably take place and, thus, accurate determination of the relative concentration of the cellular population of interest is virtually impossible. The shortcomings in the above procedures have been recognized for some time and at least one alternative has been proposed in the technical literature, articles by Caldwell, appearing in A. J. C. P., vol. 88:4 (1987) pp 447-456; and et al, A. J. C. P., Vol. 86:5 (1986), pp 600-607. The Caldwell articles, however, focus only upon one facet of this problem, which he believes to be the principle detractor from prior procedures. More specifically, Caldwell emphasizes that excessive washing of the leukocytes samples subsequent to staining and prior to analysis can introduce analytical error into the analysis, especially where leukocytes are observed in certain classes of diseased patients. Caldwell suggests adoption of a "no wash" technique to reduce processing time and analytical error which is inherent in the extensive manipulation treatment of the leukocyte sample prior to its ultimate analysis. The "no wash" technique proposed by Caldwell involves retention of the traditional techniques for preparation of an enriched lymphocyte fraction (i.e., Ficoll-Paque density gradient centrifugation, which itself contemplates multiple wash steps for removal of the Ficoll-Paque reagents). The enriched sample of lymphocytes is, thereafter, stained with a fluorochrome dye conjugated to a monoclonal antibody. Caldwell's improvement resides in the observation of enhanced fluorescence intensity in both background and positive peaks of a histogram when the unbound conjugate is allowed to remain in the sample. Caldwell attributes this observation of enhanced fluorescence intensity to his elimination of the wash steps after staining. He hypothesizes that the elimination of such wash step is less disruptive upon the stained lymphocyte fraction, thus, enabling preservation of the immunochemical bond between the conjugate and the surface markers on the cell population of interest. As is clearly evident from review of Caldwell's "no wash" procedure, the repeated trauma of mixing, vortexing and centrifugation is only somewhat reduced.
The "enrichment" of the leukocyte sample through the use of lytic reagents has not, up to now, proven to be a viable alternative to the more traditional centrifugation/density gradient techniques for partitioning the whole blood sample into its various fractions. The reason for this limitation is the inability of prior lytic reagents to effect hemolysis of the erythrocyte fraction of whole blood without also traumatizing the leukocyte fraction of the sample. These lytic reagents typically cause both gross and subtle morphological changes in the leukocyte fraction and alteration in the surface markers on such cells. In the limited number of instances where lytic reagents have been used with some measure of success, additional reagents were required to be added to the sample to protect the leukocytes from lysis by the lytic reagent, see for example, U.S. Pat. No. 4,637,986 (to Brown, et al). As is evident from the review of the Brown patent, the physiological environment of the sample is modified dramatically by the addition of both lytic reagent and the solutes which are used to protect the leukocytes from lysis by the lytic reagent. The samples prepared in accordance with Brown's reagents and technique can be subjected to analysis by flow cytometry where differentiation of the three principle sub-populations is accomplished by conventional light scattering measurements. Because of the alteration in the physiological environment of the sample, the integrity and immunochemical response of the surface markers on the leukocytes of the sample is altered as well. Thus, refinement in the analysis of the leukocyte fraction by immunochemical techniques is precluded. Moreover, where the leukocyte fraction is from a sample which is "aged" (not fresh) or a diseased patient sample, the sensitivity of the leukocytes to such harsh treatment is increased dramatically, thus, further limiting the usefulness of the Brown reagents and technique.
As is evident from the above discussion, both the physical and chemical trauma which can be effected upon the leukocyte fraction by the above procedures, is likely to damage a number of the cells within this fraction. Even more unfortunate, these procedures are more destructive of the leukocytes (i.e. lymphocytes) of disease state samples. Accordingly, the ability to analyze and monitor such disease state samples by traditional enrichment/staining techniques is severely limited. Where such analysis are to be performed, the more traditional centrifugation/density gradient enrichment protocols are preferred, since they are somewhat less disruptive of the lymphocytes than the effects of the lytic reagents.
The enhancement in accuracy and sensitivity observed by Caldwell in his "no-wash" procedure represents a step in the appropriate direction, however, further improvement is obviously necessary, particularly where relative cell population determinations are critical to an accurate analysis of the patients conditions. As is evident from the above discussion, none of the prior art techniques, even that described by Caldwell, provide a complete solution to this problem, since virtually every procedure, even those utilizing lytic reagents, occasion a statistically significant loss or destruction in one or more of the sub-populations of leukocytes and at least 1 hour total preparation time. This loss is statistically more significant in dealing with an aged sample (i.e. a sample essentially totally deficient in endogenous nutrient) and disease state samples because of the relatively higher sensitivity of the nutritionally deprived cells, and cells indicative of the disease state to lysis by physical and chemical trauma.